Engel G. Vrieling

Silicon deposition in diatoms: control by the pH inside the silicon deposition vesicle

To test the hypothesis that silicification occurs under acid conditions in the silicon deposition vesicle (SDV), the acidity of the SDV of the pennate diatoms Navicula pelliculosa (Brébisson et Kützing) Hilse, N. salinarum (Grunow) Hustedt, and Nitzschia sigma (Kützing) Smith was determined during development of new frustule valves. Cells were incubated with the weak base 3‐(2,4‐dinitroanilino)‐3′‐amino‐N‐methylpropylamine (DAMP) followed by immunocytochemical localization in whole cells and on ultrathin sections. After resupplying silicate to cells synchronized by silicon depletion, the uptake of this nutrient from the medium was the same with or without DAMP; new valves developed without morphological aberrations that could conceivably have been caused by the probe. DAMP was found in cellular compartments known to be acidic, such as vacuoles active as lysosomes, the lumen of thylakoids, and microbodies. In the nucleus and mitochondria, which are circumneutral and basic compartments, the probe did not appear. Besides its presence in acidic compartments, DAMP was specifically accumulated within the SDV during formation of new valves; during the process of valve maturation, the SDV seemed to become increasingly acidic. In control experiments using the ionophores chloroquine, valinomycin, and nigericin, the compartmental location of DAMP was clearly disturbed, resulting in a random intracellular distribution. Accumulation of the fluorescent probe rhodamine 123, which can be translocated over membranes by a reducing potential, confirmed that the SDV can translocate weak bases. The results with DAMP suggest that the pH of the SDV is important in the silicification of diatoms: It facilitates a fast nucleation and aggregation of silica particles, thus increasing the rate of formation of the mature frustules. In addition, the acidic environment might protect the newly formed valves against dissolution before completion and coverage by the organic casing prior to their secretion.

Diatom silicon biomineralization as an inspirational source of new approaches to silica production

The demand for new materials and products is still growing and the interest in naturally formed biopolymers and biominerals, such as chitin, calcium precipitates and silica is increasing. Photosynthesizing microalgae of the family Bacillariophyceae (diatoms) produce silica exoskeletons with a potential to be used in specific industrial or technological processes, they also are an excellent model in studies of silicon biomineralization. In contrast to geologically aged diatomaceous earth, the freshly prepared silica of cultured or harvested natural diatoms has been characterized insufficiently with respect to the properties (e.g. purity, specific surface area, porosity) required for technological and industrial application. In this contribution we summarize aspects of cellular processes that are involved in silicon biomineralization of diatoms and the current knowledge of the characterization of diatomaceous silica, following methods used for synthetically derived silica-based materials.

Immunofluorescence in phytoplankton research: Applications and potential

During the last decade, immunofluorescence has become an important tool in studies of the systematics, biogeography, physiology, and ecology of freshwater and marine phytoplankton. The technology originated in the medical sciences (e.g. Coons et al. 1941, 1955) and was gradually applied to soil bacteria (Fliermans et al. 1974, Josserand and Cleyet-Mare1 1979) and bacteria from aquatic environments (Fliermans and Schmidt 1977, Gates and Pham 1979, Ward and Perry 1980, Baker and Mills 1982, Dahl and Laake 1982, Yoshioka et al. 1982, Ward and Carlucci 1985, Currin et al. 1990). Applications of immunology to phytoplankton originated over two decades ago with the innovative studies of Bernhard et al. (1969), who attempted to differentiate phytoplankton species using antibodies. Modern immunological studies of phytoplankton are more diverse in their applications, but the same principles of using antibodies to label and visualize target cells or cellular constituents still apply. Coupled with the power and sensitivity of fluorescence, immunological techniques open the door to physiological and ecological investigations that formerly were not possible. This is especially true in studies that focus on a single species within a complex field assemblage of organisms and detritus. Applications of immunofluorescence include identification and enumeration of phytoplankton species, localization and quantification of cell constituents (enzymes, toxins, structural proteins, and polysaccharides), and labeling of cells for better quantification of grazing rates and mixotrophic or heterotrophic potential. Given the rapid pace of development in this field, it seems appropriate at this time to review the methods, applications, and potential of immunofluorescence as a tool in phytoplankton research, in hopes that such an overview will help to sustain and focus future investigations.

Salinity-dependent diatom biosilicification implies an important role of external ionic strength

The role of external ionic strength in diatom biosilica formation was assessed by monitoring the nanostructural changes in the biosilica of the two marine diatom species Thalassiosira punctigera and Thalassiosira weissflogii that was obtained from cultures grown at two distinct salinities. Using physicochemical methods, we found that at lower salinity the specific surface area, the fractal dimensions, and the size of mesopores present in the biosilica decreased. Diatom biosilica appears to be denser at the lower salinity that was applied. This phenomenon can be explained by assuming aggregation of smaller coalescing silica particles inside the silica deposition vesicle, which would be in line with principles in silica chemistry. Apparently, external ionic strength has an important effect on diatom biosilica formation, making it tempting to propose that uptake of silicic acid and other external ions may take place simultaneously. Uptake and transport of reactants in the proximity of the expanding silica deposition vesicle, by (macro)pinocytosis, are more likely than intracellular stabilization and transport of silica precursors at the high concentrations that are necessary for the formation of the siliceous frustule components.

Chattonella and Fibrocapsa (Raphidophyceae): First observation of, potentially harmful, red tide organisms in Dutch coastal waters

Species of the potentially toxic and red-tide-forming marine-phytoplankton genera Chattonella and Fibrocapsa (Raphidophyceae) were observed for the first time in 1991 in samples taken in Dutch coastal waters; they were again recorded and enumerated in the following years. Chattonella spp. cell numbers varied with the season, with a maximum in May or June in the Dutch Wadden Sea. Cell numbers of Chattonella and F. japonica Toriumi et Takano were up to 6.0 . 10(3) cells . dm(-3) in the Dutch Wadden Sea, except at one station in June 1993 when over 10(4) cells . dm(-3) Chattonella were counted. In May 1993, a minor bloom (over 2.0 . 10(5) cells . dm(-3)) was observed at a station in the southern central North Sea, 100 km northwest of the island of Terschelling. The potentially neurotoxic species Chattonella marina (Subrahmanyan) Hara et Chihara was identified and discriminated from morphologically related species within the class of Raphidophyceae by immunofluorescence. F. japonica could only be clearly identified in live samples; in fixed samples cell morphology was severely affected. The identification of this species was supported by the presence of mucocysts, structures that can be observed readily by optical and electron microscopy.

Nanoscale uniformity of pore architecture in diatomaceous silica: a combined small and wide angle x‐ray scattering study

Combined small and wide angle X‐ray scattering (SAXS and WAXS) analysis was applied to purified biogenic silica of cultured diatom frustules and of natural populations sampled on marine tidal flats. The overall WAXS patterns did not reveal crystalline phases (WAXS domain between 0.07 to 0.5 nm) in this biogenic silica, which is in line with previous reports on the amorphous character of the SiO2 matrix of diatom frustules. One exception was the silica of the pennate species Cylindrotheca fusiformis Reimann et Lewin, which revealed wide peaks in the WAXS spectra. These peaks either indicate the presence of a yet unknown crystalline phase with a repetitive distance (d‐value ≈0.06 nm) or are caused by the ordering of the fibrous silica fragments; numerous girdle bands. The SAXS spectra revealed the size range of pores (diameter d between 3.0 and 65 nm), the presence of distinct pores (slope transitions), and structure factors (oscillation of the spectra). All slopes varied in the range of −4.0 to −2.5, with two clear common regions among species: d < 10 nm (slopes –4, denoted as region I and also called the Porod region), and 10.0 < d < 40.0 nm (slopes −2.9 to −3.8, denoted as region II). The existence of these common regions suggests the presence of comparable form (region I) and structure (region II) factors, respectively the shape of the primary building units of the silica and the geometry of the pores. Contrast variation experiments using dibromomethane to fill pores in the SiO2 matrix showed that scattering was caused by pores rather than silica particles. Electron microscopic analysis confirmed the presence of circular, elliptical, and rectangular pores ranging in size from 3 to 65 nm, determining the structure factor. The fine architecture (length/width ratio of pore diameters) and distribution of the pores, however, seemed to be influenced by environmental factors, such as the salinity of and additions of AlCl3 to the growth medium. The results indicate that diatoms deposit silica with pores <50 nm in size and are highly homologous with respect to geometry. Consequently, it is suggested that in diatoms, whether pennate or centric, the formation of silica at a nanoscale level is a uniform process.

Location and expression of frustulins in the pennate diatoms Cylindrotheca fusiformis, Navicula pelliculosa, and Navicula salinarum (Bacillariophyceae)

A detailed immunocytochemical and biochemical study of the location and expression of frustulins, a family of proteins associated with the frustules of diatoms, has been performed for Cylindrotheca fusiformis Reimann et Lewin, Navicula pelliculosa (Brébisson et Kützing) Hilse, and Navicula salinarum (Grunow) Husted. Immunocytochemistry revealed that frustulins, which share homologous epitopes but are different in size, were predominantly located in the organic casing. Based on timed immunolocalization experiments and Western blotting analysis of cell extracts obtained sequentially after repleting silicate to Si‐synchronized cells, the continuous presence of the frustulins in the mature and parental organic casing of the examined species was observed. The frustulins of N. pelliculosa appeared as proteins similar to those of C. fusiformis, sharing identical epitopes. The extractions, however, yielded a markedly lower abundance of frustulins in N. pelliculosa. Peak concentrations of extracted frustulins appeared to be expressed just ahead of the silicification process in C. fusiformis, whereas the level of expression in N. pelliculosa increased along with maturation of the new valves. For N. salinarum, the presence of the frustulins could not be confirmed properly by Western blotting, most probably because of the small sample volumes, inefficient extraction, and a lower amount of homologous frustulins in the casing of this species. It is concluded that the frustulins of these species are not associated with the silicalemma of the newly formed silica deposition vesicles and therefore do not seem to be involved in the silicification process itself. Overall, the results imply a structural role of the frustulins in the casing of diatoms rather than a regulation of the silicification process.

Growth and silica content of the diatoms Thalassiosira weissflogii and Navicula salinarum at different salinities and enrichments with aluminium

The dependence of the cellular (biogenic) and frustule-associated (mineralized) silica content of the diatoms Navicula salinarum and Thalassiosira weissflogii on salinity and aluminium conditions was studied in order to make it possible to manipulate silicification in vitro and maximize it to levels required for physico-chemical frustule characterization by physisorption, X-ray scattering analysis and NMR, which is our ultimate objective. Enrichments with AlCl3 increased growth and the final cell density of the pennate N. salinarum, but not of the centric species T. weissflogii. Aluminium additions did not, however, result in a significant increase in the biogenic or mineralized silica content per cell and could not be detected in the silica matrix of the frustule. In contrast, lowering the salinity from 28 practical salinity units (PSU) to 20 and 15 resulted in a significant increase in the biogenic silica content per cell of both species, which is in line with an increase in density of the chemically de...

Monitoring rapid valve formation in the pennate diatom Navicula salinarum (Bacillariophyceae)

After each division of a diatom cell, a new siliceous hypovalve is formed inside the silica deposition vesicle (SDV). We present the sequence of this early formation of the new valve in the pennate marine diatom Navicula salinarum (Grunow) Hustedt, visualized by using the fluorescent probe 2‐(4‐pyridyl)‐5‐((4‐(2‐dimethylaminoethylamino‐carbamoyl)methoxy)phenyl)oxazole (PDMPO). Our observations confirm that two‐dimensional expansion of the growing valve is a rapid process of no more than 15 min; three‐dimensional completion of the valve appears to be slower, lasting most of the time valve formation takes. The results are relevant to studies of the timing of molecular processes involved in valve formation (i.e. the bio‐ and morphogenesis of the SDV) in relation to uptake and transport of silicic acid. Use of this probe helps us to identify specific developmental stages for further detail analysis of diatom basilica formation, which eventually could lead to obtaining enriched SDV fractions.

Identification of a domoic acid-producing pseudo-Nitzschia species (Bacillariophyceae) in the Dutch Wadden sea with electron microscopy and molecular probes

Biological monitoring in the Dutch Wadden Sea between November 1993 and July 1994 revealed Pseudo-nitzschia-like pennate chain-forming diatom species with cell numbers ranging from 102 to 105 per litre. Cultured isolates and field samples were examined by electron microscopy, which revealed the majority to be Pseudo-nitzschia pungens. This species dominated over other diatoms in the phytoplankton population during November 1993 and at the end of June 1994. At the beginning of June 1994, P. fraudulenta was also present; occasionally, P. delicatissima was observed. One isolate showed the characteristic morphology of P. multiseries. Species-specific polyclonal antibodies and large-subunit (LSU) rRNA-targeted oligonucleotides for North American strains of P. multiseries and P. pungens applied to the European isolates, confirmed species designations based on electron microscopy. The isolate of P. multiseries from the Dutch Wadden Sea produced domoic acid; after 55 days of growth about 19 pg per cell was measur...